Confocal imaging offers a number of advantages over conventional microscopy for quantitative imaging, including improved image contrast, resolution and limited depth of field to support optical sectioning or quantitative three dimensional spatial sampling. Conventional confocal imaging systems incorporate several basic functional subsystems in order to achieve the generally cited advantages of confocal imaging. These functional subsystems include: a system for scanned illumination; a spatial filter or aperture in the focal plane of the detector to reject out-of-focus light; and imaging optics to bring the illumination and imaging paths into focus at the same location.
A conceptually simple prior art confocal microscope for transmission imaging involves light from a light source being transmitted through a pinhole and brought to focus within a sample volume by imaging optics. Light emerging from the sample is brought to focus on a pinhole aperture by a second set of imaging optics. This aperture rejects most of the light that did not pass through the confocal point in the sample volume, thereby reducing image contributions from light scattered or refracted from other locations within the medium.
The detector for such a system typically consists of a single channel device such as a photodiode or a photomultiplier. In order to collect an image, it is necessary to scan the illumination and detection subsystems in a coordinated manner.
However, the technical requirements for coupled electromechanical scanning to preserve the required optical alignment between illumination and imaging pathways are formidable. Most practical embodiments of a transmission can significantly enhance the speed, flexibility and ease of use confocal system utilize mechanical scanning of the sample so that the imaging elements do not move.
The typical configuration for confocal imaging in reflected light or fluorescence modes employs the same imaging optics and often the same pinhole aperture for both the illumination and imaging paths. This arrangement simplifies optical alignment and facilitates optical scanning, either by mechanical movement of the aperture, or by shifting the image of the aperture by mirrors in the optical path. In most designs, the scanning of the illumination path and the "descanning" of the imaging path are accomplished with the same set of mirrors. Image acquisition in most existing systems is slow, a fact which complicates preliminary inspection of a sample and the focusing of the scanned image.
Spinning disk confocal systems are a useful implementation of the reflected light or fluorescence modes described above. In this type system, the scanning and aperture functions are embodied in an opaque disk containing a pattern of optically transmissive pinholes arranged in a spatial array so that during a revolution of the disk, the entire sample image plane is illuminated. The disk is rotated at a high speed, with each pinhole serving as the aperture for both the illumination and the imaging paths. Because a number of locations within the sample are scanned in parallel, the detector consists of a spatially resolved area detector such as a video camera, although direct viewing by eye is also possible. These systems can significantly enhance the speed, flexibility and ease of use of confocal microscopy, but they typically suffer from low sensitivity because most of the incident light is absorbed by the opaque regions of the disk, and from problems associated with reflection from the disk surface. These systems also are relatively bulky, and the gyroscopic nature of the spinning disk inhibits their use in compact portable imaging systems such as endoscopes.
Another complementary strategy for high performance confocal imaging is to use an illumination source configured as a line instead of a point, with a slit aperture. This allows parallel detection using a specialized detector such as a linear CCD, considerably reducing imaging time. This performance enhancement comes at the expense of some degree of compromise in the image quality. This is because, in a line scan configuration, out-of-focus light that happens to fall onto the slit aperture contributes to the image. However, in practice, the degradation of confocal image quality is not unacceptably severe. This type system does produce a brighter image, although the additional light is presumed to be non-confocal. Such systems are considerably simpler and faster than systems used for point scanned illumination, since the pattern of illumination is scanned in only one dimension. Other approaches to confocal scanned illumination in principle offer even higher performance. One existing instrument scans a laser in two dimensions, using a galvanometrically driven mirror for one dimension and an acousto-optic beam steering device for the second. This system achieves confocal image rates faster than standard video. However, in fluorescence mode the emission wavelength shift causes image light to be descanned improperly by the acousto-optic device. The confocal image quality of this device is comparable to that of a linescan confocal imager.
Another strategy for high performance confocal imaging is to dispense with mechanical scanning altogether, and to use acousto-optic devices for scanning a laser in both dimensions. In one such design, the detector is based on an image dissector videcon, a television detector tube that allows random access readout of the tube photocathode at user defined coordinates specified by a pair of analog voltages. Readout position can be adjusted to precisely match a peak of illumination in the imaging detector. However, as with most other tube-based video detectors, the image dissector has been rendered obsolete by the advent of solid-state video devices. A commercial version of this type of confocal imager has not been produced. Because optoacoustic devices operate by modulating the index of refraction of a transmissive element, the devices are of little utility for steering white light. Even for laser applications, the beam displacement varies as a function of wavelength, and polychromatic light is dispersed, creating problems for confocal imaging.
A number of strategies for electronically programmable illumination have been explored, although until recently none has proven practical. An early strategy was to use the spot from a CRT as a moveable illumination source for scanned microscopy. However the intensity proved to be too low for practical microscopic imaging. The advent of video projectors based on LCD devices in the late 1980s stimulated the development of prototype scanning systems. However, the size, inefficiency and low contrast of the devices precluded the development of a practical imager. Subsequent development of miniature LCDs has addressed some of these problems, although pixel quality was a problem in early devices. In addition, LCDs achiever gating of transmitted illumination based on polarization, limiting their efficiency for illumination and placing constraints on their use as an imaging aperture, particularly for fluorescence imaging.
New technologies based on micromechanical devices appear to address many of the concerns noted with earlier prototype systems based on LCD and similar devices. Digital Light Processing (DLP) technology developed by Texas Instruments employs an array of microscopic mirrors that can tilt back and forth between two limit positions based on the state of an associated Random Access Memory (RAM). Light from a collimated illumination source is reflected into the optical path or is dumped into a light sink. Devices presently available are optimized for video projection applications, requiring a digital video source to drive the array, and producing the perception of analog intensity by rapidly flickering individual mirrors. However, it is possible to use such an array for binary illumination driven by direct access of the device frame RAM by a host computer.
A recent patent issued to Krause (U.S. Pat. No. 5,587,832) describes an application of such devices for confocal imaging. The patent describes the use of a novel microshutter array; however, the fundamental strategy is compatible with the use of existing micromirror technology. One array is used as a source for programmable scanned illumination. Another array (or the same one) is used as a programmable aperture array for an associated 2D-area detector, such as a CCD. Such an imaging configuration is relatively simple to realize for reflected light or fluorescence modes employing a single array for illumination and imaging. However, the requirement to precisely align all of the corresponding shutter pairs for a transmitted light measurement is likely to prove a significant technical challenge. Although this system employs an alternative technology, the approach is not different in principle from spinning disk confocal systems. A physical aperture array is used to produce a temporal sequence of complementary spatial patterns of illumination. In the imaging pathway, a correlated physical aperture array is employed to reject out-of-focus light.
A practical problem with this approach is its inefficient use of illumination light. In order to minimize overlap between the point spread functions of adjacent illuminated pixels, these sites must be separated in both dimensions of the illumination array. A comparatively small fraction of the pixels are on at any given time during scanning. For example, if every fifth pixel in x and y are turned on, only 1/25 of the available light is used in any given image. This means that the pattern must be shifted 25 times, and that 25 component images are acquired to completely sample the image field. In practice, this approach is acceptable for many transmitted light applications, and for reflected light imaging of highly reflective samples. However, the inefficiency of this device is a serious handicap for light limited applications such as fluorescence imaging.
In the present invention, an alternative method for producing a regular array of scanned illumination points has been developed. Microlens arrays that consist of many small lens elements in a close packed array now are commercially available. Existing arrays have as many as 256.times.256 elements. The pitch between adjacent elements (i.e. the center to center spacing) is often 200 microns or less. If collimated light is passed through such a lens array, an array of spots is produced in a plane displaced from the microlens array by the focal length of the lens elements. The effective aperture of the array is large, although the acceptance angle of individual elements may be limited. Typically, greater than 95% of incident light may be transmitted by such an array. These arrays are small (mm to cm) and lightweight, and can be mechanically scanned by range of devices including galvanometers, stepping motors, piezo-ceramic devices, and other actuators. For 2-dimensional scanning, it only is necessary to translate the microlens array over distances comparable to the pitch length, with incremental displacements dependent on the desired resolution of the scan. To simplify mechanical requirements for scanning a regular microlens array, the array can be tilted relative to the axis of translation. This allows scanning in one dimension to achieve resolution of less than the pitch length in both dimensions. However, the same number of incremental displacements is required to achieve the same resolution.
Thus, a number of practical solutions for scanned illumination suitable for use in confocal imaging have been developed, including the novel method described above. However, most methods for true confocal imaging demonstrated to date, in addition have required a physical imaging aperture. This is often a pinhole in a conjugate focal plane of the imager. In some designs, the imaging pinhole is replaced with a concave (diverging) lens, and an adjustable iris-type aperture. This allows collection of more light by graded adjustment of the aperture, at the expense of degraded confocal performance. In another patented design, an optical fiber replaces the pinhole aperture used for illumination and detection. This approach greatly simplifies the linkage between a conventional microscope and confocal illumination and detection subsystems. In most practical designs, the imaging aperture is the same as the illumination aperture, or is mechanically coupled to it. A notable exception is the use of an electronically driven aperture array as disclosed in the Krause patent, but, even in this case, a physical aperture is employed.
The confocal imager employing an image dissector vidicon is an example of a virtual aperture. No physical device is used to limit the light reaching the detector array, but the effects of such an aperture are simulated by the method of readout of the detector. The use of an analog image tube is an important feature of this approach, since it allows the region sampled by the image dissector tube to be precisely aligned with the image of the illumination spot.
It also is possible to construct a virtual aperture using a modern area detector such as a CCD. Pixels in such an imager correspond to discrete elements within an array. Because each detector element accepts light from a limited region of the focal plane, it serves as a small fixed aperture.
A patent issued to Batchelder et al. (U.S. Pat. No. 5,510,894) describes a simple form of virtual confocal aperture for Raman spectroscopy accomplished by selective binning and readout of the photoreceptor array, or by equivalent postprocessing procedures. This strategy produces an effect analogous to a pinhole in a conventional spatial filter used for confocal imaging. Although specific aspects of this method are rather specific for Raman spectroscopy, the general ideas can be extended for other forms of spectroscopy. The Batchelder et al. patent also describes a simple extension of the method for spatially resolved spectroscopy or spectral imaging, implemented by placing the sample on a scanning table, and sequentially applying the same virtual aperture to the series of point or line spectra arising from the raster scan of the sample. This is a slow, awkward system that employs expensive apparatus, and which places severe constraints on its application. Such a system is not suitable for imaging of dynamic processes (or even real-time imaging of slow processes), and it requires that the sample be small and moveable. This often is not the case in macroscopic or endoscopic applications.
The Batchelder et al. patent does not anticipate the use of spatially scanned illumination with a fixed sample and imager to achieve a spectrally resolved image, nor does it anticipate that many points might be illuminated in parallel to greatly increase the efficiency of confocal spectral imaging. In the Batchelder et al. system, the imaged point or spectrum occurs at the same position on the detector array regardless of the position of that point on the sample. The processing methods described by Batchelder et al. do not deal with several complications posed by the inconsistent sampling of the continuous image by the sensor array relative to a scanned illumination point source or illumination array. These are significant extensions to the computational method which are necessary to construct a virtual aperture array suitable for use with scanned illumination schemes.
Although the present invention is based on the same principles as the prior art confocal imaging systems, it realizes the necessary functional subsystems in a novel and beneficial way. It enables a number of possible applications and produces numerous advantages.
It is therefore an object of the present invention to provide a confocal imaging apparatus sufficiently compact to allow its use in medical applications.
It is a further object of the present invention to provide confocal imaging apparatus having high sensitivity, speed and dynamic range in a flexible and cost effective implementation.
It is a still further object of the present invention to provide confocal imaging apparatus that can produce a complete spectrum for each image pixel.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.